This project will result in the development of new cell-screening technologies based on single molecule detection. Based on previous results using fluorogenic caspase probes, cellular processes that are normally observed after several hours by conventional tools can be detected in less than one hour. We will extend these results to develop a microfluidic cell-scanning system that can assay fluorogenic probes in an automated fashion. Using single molecule fluorescence, several parameters can be determined simultaneously; including fluorescence burst intensity (counting molecules), fluorescence recurrence time (diffusion-corrected concentration), and fluorescence correlation time (fluorescence correlation spectroscopy). New fluorogenic protease probes will be developed that feature red-laser excitation. We will use apoptosis as our test model, and develop caspase-specific probes. Upon proteolytic cleavage, the fluorogenic probe will be converted to a fluorescent molecule with an emission near 660-690 nm. This region of the spectrum is free from cell autofluorescence and will allow for even faster detection. The result will be a method for assaying a biochemical system, in living cells, with high temporal resolution. In an effort to improve the cell throughput of our existing system, an automated microfluidic scanning and data analysis system will be developed. Cells will be scanned past the laser beam using segmented flow and laser trapping. Single molecule data will be subsequently obtained. A decision-making module in the software will compare measured parameters to control values and classify cells according to protease activity. The end result of this project will be an instrument capable of multiparameter fluorescence detection, single molecule sensitivity, and automated cell throughput. While apoptosis and caspase activity are chosen as a test case, this instrumentation and methodology can be adapted easily to other biomedical problems. PUBLIC HEALTH RELEVANCE (provided by the applicant): We will develop new molecular probes, instrumentation, and methods to study intracellular protein-substrate interactions with high temporal resolution. This methodology will allow biological processes to be studied with higher throughput and sensitivity.